Protein quantitation using mass spectrometry requires integration of sample preparation, instrumentation, and software. Strategies to improve sensitivity and comprehensiveness generally require large sample quantities and multi-dimensional fractionation, which sacrifices throughput. Alternately, efforts to improve the sensitivity and throughput of protein quantification necessarily limit the number of features that can be monitored. For this reason, proteomics research is typically divided into two categories: discovery and targeted proteomics.
Discovery proteomics experiments are intended to identify as many proteins as possible across a broad dynamic range. This often requires depletion of highly abundant proteins, enrichment of relevant fractions, and fractionation to decrease sample complexity.
Quantitative discovery proteomics experiments often utilize isotopic labeling methods, such as labeling with isobaric tags and isotope-coded affinity tags (ICAT) to quantify the proteins. Isobaric tags, which are commercially available in the form of tandem mass tags (TMT) reagents (sold by Thermo Fisher Scientific) and isobaric tags for relative and absolute quantitation (iTRAQ) reagents (sold by AB Sciex), have been repeatedly demonstrated as a facile means of obtaining relative protein quantitation in both small and large scale proteomics studies while providing the powerful capability of simultaneously comparing up to ten different treatments, time points, or samples. These protein labeling strategies incorporate isotopes into proteins and peptides, resulting in distinct mass shifts but otherwise identical (among peptides labeled with different isotopomers of a reagent set) chemical properties. This allows several samples to be labeled and combined prior to processing and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This multiplexing reduces sample processing variability, improves specificity by quantifying the proteins from each condition simultaneously, and requires less liquid chromatography-mass spectrometry (LC-MS) and data analysis time.
While there is some cost associated with the mass tagging kits, tens of thousands of proteins can be identified and quantified across multiple samples (representing, for example, different disease states) in a highly efficient multiplexed fashion with a single LCMS experiment. Numerous high importance and significantly regulated protein targets of interest often emerge from these studies and become the subject of future routine analyses using highly sensitive and robust targeted techniques, without the added expense or steps of isobaric labeling.
Targeted proteomics experiments are typically designed to quantify less than one hundred proteins with very high precision, sensitivity, specificity and throughput. Targeted MS quantitation strategies use specialized workflows and instruments to improve the specificity and quantification of a limited number of features across hundreds or thousands of samples. These methods typically minimize the amount of sample preparation to improve precision and throughput.
Targeted quantitative proteomic workflows typically involve protein denaturation, reduction, alkylation, digestion and desalting prior to LC-MS/MS analysis on a mass spectrometer instrument capable of quantifying peptides by monitoring specific mass windows for peptides of interest, fragmenting the isolated peptide(s), and then quantifying several fragment ions that are specific for the peptide of interest. This acquisition strategy, of which selective reaction monitoring (SRM) is a common example, together with chromatographic retention time information, provides very high sensitivity, specificity, dynamic range and throughput.
Unfortunately, current workflows suffer from a severe disconnection between isobarically labeled discovery quantitation and routine targeted label-free quantitation of significant peptides. As the isobaric labeling alters not only the mass of the peptide but also its charge (when ionized) and hydrophobicity, targeting a highly regulated peptide from an isobaric experiment is not straightforward. Yet, scheduling of analytes using their retention time is important for reproducible quantitation when the list of targeted peptides grows long. This often necessitates an intermediate validation step using a label-free quantitative technique such as data independent acquisition (DIA) or MS1 based discovery quantitation which can be costly in terms of performance, sample, instrument time, and reagents. While it is possible to use label-free discovery proteomics and then directly transition to targeted routine quantitation of key peptides, multiple serial analyses must be performed to deliver the same information acquired in a single multiplexed isobaric labeling experiment.
What is needed is a method and system for translating directly from multiplexed isobaric labeling discovery quantitation to routine label-free targeted quantitation without an intermediate validation step.